1. Field of the Invention
The present invention relates to a biomarker for Alzheimer's disease (AD) comprising a complex of an Aβ amyloid peptide and a cell or a cellular membrane and to a non-invasive method for diagnosing Alzheimer's disease or monitoring its development or progression using this biomarker.
2. Description of the Related Art
Alzheimer's disease (AD) is a progressive neurodegenerative disorder resulting in loss of memory and cognitive function that disrupts work, hobbies and daily life. This disease is a dementia, progressing over several years from very mild to severe stages. About 35.6 million people worldwide are presently living with dementia; this number should increase to 65 million by 2030 and 113 million by 2050. Indeed, AD is the most common cause of dementia representing 60% to 80% of all dementia cases in adults aged 65 and over (Brodaty et al., 2011), thus imposing significant burdens on the affected individuals and their families as well as economic and social costs on medical and healthcare resources in both developed and emerging countries.
Alzheimer's disease (“AD”) is a progressive disease. It is now well recognized that biochemical changes preceding AD may be present in an affected individual up to 20 years before clinical symptoms develop and that physiopathological processes associated with the disease begin many years before the clinical diagnosis is set.
Disease pathology is associated with increased deposits of β-amyloid (Aβ) plaques in the brain as well as with increases in the intracellular aggregates of Tau protein that form neurofibrillary tangles in neurons (Marcus et al., 2011, Gotz et al., 2012). This last event is associated to a progressive neuronal loss.
Currently, there is no single test to detect Alzheimer's disease. Usually only a “potential” diagnosis is made based on a combination of clinical examination, neuropsychological tests, and brain imaging that is conducted over a series of weeks or months. Sometimes detection of biomarkers in cerebrospinal fluid is also performed but this requires a lumbar puncture to obtain a biological sample for assay. Lumbar punctures are often avoided because they are painful and risky to the tested patient. Unfortunately, an accurate diagnosis of AD was often only provided by autopsy, until now.
Conventional tests attempting to diagnose AD faced two important limitations: they lacked the sensitivity necessary for early detection of AD and were unreliable. Diagnosis of Alzheimer's disease was often assessed very late in its progression, for example, 3 to 4 years after a patient's first complaint, thus preventing the initiation of early care and delaying therapy or proper management of the disease and its associated disabilities. Conventional tests were also unreliable since they lacked the specificity and sensitivity necessary to identify subjects having AD, for example, tests measuring amyloid proteins in blood plasma yielded different results depending on the amount of binding of amyloid proteins to blood components or depending on the time of day when a blood sample was obtained. These conventional tests lacked an easily accessible, sensitive and specific biomarker for AD.
This was a major impediment and bottleneck to developing reliable and rapid biochemical tests for the pathologies associated with Alzheimer's disease. Another impediment was the identification of a biomarker that did not require invasive sample collecting, such as a spinal tap.
The lack of such an accessible, sensitive and specific biomarker that could be validated by cellular, animal model, pre-clinical models, and human testing impeded the development of therapies and drugs for AD or for the pathological processes triggering AD or involved in the progression of AD.
Pathological phenomena associated with the development of Alzheimer's disease were known. However, knowledge of these phenomena did not provide accessible, sensitive and specific biomarkers for AD. The underlying neurodegenerative mechanism of AD involves several interacting processes: neuronal death, oxidative stress, abnormal protein processing particularly beta-amyloid production (Aβ) and tau and mitochondrial dysfunction. These processes result in the characteristic accumulation of beta-amyloid plaques, neurofibrillary tangles and synaptic loss, ultimately leading to cerebral atrophy (Selkoe et al. 2002; Terry R D et al., 2006). More precisely the amyloid precursor protein (APP) is hydrolysed to give beta-amyloid peptides (Aβ) of various lengths the prominent species having 42 or 40 amino acids. These highly hydrophobic peptides display a beta sheet structure and are the major constituents of the amyloid plaques in the central nervous system. The Tau protein is associated to the microtubule network inside nerve cells. During the disease course the Tau protein is hyperphosphorylated, it dissociates from the microtubules and forms neurofibrillary tangles.
For research purposes, compounds that specifically interacted with amyloid plaques were developed. When properly labeled with radioisotopes these compounds allowed in vivo imaging of amyloid deposits in human brain and in animal models (Jack et al., 2011; Poisnel et al 2012). However, this methodology required radiolabelling and positron emission tomography, thus restricting its use to investigational or research purposes only. It did not provide a practical way to assess or diagnose a patient for AD. Moreover, brain amyloid deposits are not directly correlated to disease progression (Chuang et al., 2012; Choi et al 2012). Although characteristic neuropathologic changes are described (e.g., accumulation of beta-amyloid plaques and neurofibrillary tangles that begin in the entorhinal cortex and medial temporal lobe and extend gradually to the entire neocortex; Braak et al., 1991), the cause of the disease was not clear (Hardy et al., 2002).
Pathogenesis was also associated with the accumulation of the highly amyloidogenic peptide Aβ1-42. More recently increasing evidence accumulated that the cytoxic properties of soluble Aβ peptides (either as monomers or more probably as oligomers) were at the origin of the observed neurodegenerating process (Klein et al, 2001, 2002, 2004; Hardy et al., 2002; Selkoe et al., 2002, 2008). According to these observations the hypothesis was made that the measurement of soluble monomers or oligomers of Aβ would be useful to detect AD and possibly to predict the disease. However, use of these amyloid peptides and their oligomers proved problematic since their levels varied at different times of day and their hydrophobicity caused them to bind to blood components and thus varied their concentrations in blood plasma or serum.
Among available biochemical assays the most commonly used is performed on cerebro-spinal fluid samples and involves immunochemical measurement of amyloid peptides (Aβ1-42 and Aβ1-40), of the protein tau and its hyper-phosphorylated form (Frankfort et al., 2008; Funke et al., 2009; Gabelle et al., 2010; Rosén et al 2012). This assay however was hindered by the need of a spinal tap to obtain the sample and this procedure is expensive and risky for the patient. These prior assays did not provide a way to non-invasively assess Alzheimer's disease since they required invasive sample collection procedures usually from the central nervous system, such as the collection of CSF by spinal tap as opposed to more accessible samples collected by less risky methods such as the collection of peripheral blood, urine or mucosal secretion.
As mentioned above, blood plasma and serum were known to contain substantial amounts of soluble amyloid peptides and tau protein. Numerous publications described attempts to measure such soluble peptides in blood (for a review see Frankfort et al., 2008). However, attempts to develop a non-invasive assay by measuring Aβ peptides in blood or serum were unsuccessful in producing a reliable and repeated assay. It was difficult to obtain reliable measurements of the amounts or concentrations of Aβ peptides in blood plasma or serum because Aβ peptides are hydrophobic and interact with circulating plasma proteins (Kuo et al. 2000). The repeatability and reliability of assays of Aβ concentration in blood plasma was poor due to observed circadian variations of plasmatic Aβ concentration in rats and in human subjects, which made it difficult to accurately measure Aβ concentration with a diagnostic significance.
In addition to Aβ monomers, Aβ oligomers (also called ADDLs) were considered as potentially relevant biomarkers of Alzheimer's disease (Haes et al. 2005; Georganopoulou et al. 2005) and monoclonal antibodies were developed for diagnostic and therapeutic purposes (Funke, et al., 2009; U.S. 2006/0228349 A1; and WO 2006/014478 A1). However, the reliability and significance of results based on measurement of Aβ oligomer concentration in blood plasma was poor for reasons similar to those for Aβ monomers. The accurate measurement of soluble Aβ oligomers was hindered by their interaction with circulating proteins in blood serum and no correlation was established between plasma or serum concentration of Aβ oligomers and the state of AD.
Other blood tests proposed for assessment of AD included multiparametric tests based on genomic, transcriptomic or proteomic analysis of blood lymphocytes. These multiparametric tests were performed on lymphocytes and relied upon assessment of 50 to even more than 150 different parameters. The number and complexity of these assessments and the often complicated technologies used for such measurements make these tests expensive and require the use of special equipment. Moreover, notwithstanding the complexity and expense, clinicians were reluctant to use such methods for detecting AD among mixed forms of dementia since published data show that the specificity and the sensitivity of these tests (below 78%) were not high enough for such a purpose. Consequently, when attempting to definitively diagnose a patient as having AD, the tests did not meet the expectations and requirements of many clinicians.
Generally, the cellular effects of Aβ application to living cells have been observed with Aβ concentrations in the 0.1 to 1 micromolar range (Arispe et al. 2010; Cizas et al., 2011; Ray et al 2011). These concentrations however are not physiological since soluble Aβ peptides concentrations encountered in healthy control or in pathologic tissues are in the range of 0.1 to 10 nanomolar (Neniskyle et al., 2011; Nag et al., 2005 and 2011). It was previously observed that nanomolar Aβ peptide application had no effect on the Ca2+ intracellular concentration in living cells (Demuro et al., 2005; Chin et al., 2006; Bezprozvanny et al., 2009; Demuro et al., 2010). Recent studies suggested that several conformational forms of Aβ peptide can bind to cellular membrane (Suwalsky et al 2009). However no link either chemical or functional had previously been observed between the different forms of Aβ bound to the cellular plasma membranes (Bateman et al., 2009).
An immunological determination of beta-amyloid peptide on blood cells membranes as a biomarker of AD was described by Pesini et al., 2009. In this study, the detection of Aβ was performed by an immunochemical method on a limited number of samples from healthy controls and AD patients. However, no significant discrimination was observed between AD patients and controls. This immunochemical method was not able to discriminate AD patients from healthy control individuals and did not employ the biomarker discovered by the inventors and disclosed herein. Indeed the primary use of an antibody directed against beta-amyloid peptide as performed by Pesini et al (2009) does not allow to detect the biomarker of the present invention, which is the direct detection of the link between high affinity and low affinity binding modes of the beta amyloid peptide to the cellular membrane.